Thrombin is a multifunctional plasma protease generated in the blood stream through activation of its inactive precursor, prothrombin (also known as factor II). Prothrombin is itself activated by the action of activated factor X (factor Xa). One of the main functions of thrombin in blood plasma is to convert soluble fibrinogen into insoluble fibrin in the final step of the blood clotting cascade. The fibrin monomers produced by the action of thrombin cluster together and are then cross linked to form a blood clot. Thrombin is used in therapy both alone and in combination with fibrinogen in the so-called fibrin sealants to achieve haemostasis, to seal wounds and for the controlled adhesion of tissue.
For all clinical applications, it is important to have highly pure thrombin in order to minimise any undesirable side effects resulting from, for example, the presence of other proteases or clotting factors, or other contaminating components. In addition, it is highly desirable that thrombin for clinical use, in particular if it is derived from human or animal sources, is treated to inactivate any blood-borne viruses which may be present, for example hepatitis viruses or HIV. Various methods of virus inactivation are known in the art, including pasteurisation, dry heat treatment and solvent-detergent treatment (Pathogen Inactivation of labile blood products, Council of Europe Expert Committee in Blood Transfusion Study Group on Pathogen Inactivation in Labile Blood Products, Transfusion Medicine, 2001, 11, 149-175). It is also desirable that other pathogens are removed or reduced, for example the causative agents of Transmissible Spongiform Encephalopathies (TSE), currently believed to be prions.
Dry heat treatment is known to be effective for the inactivation of both enveloped and some non-enveloped viruses, whilst solvent-detergent treatment is known to be effective for the inactivation of enveloped (i.e. lipid coated) viruses such as hepatitis B. Solvent-detergent treatment is now a commonly used method for virus inactivation of blood products intended for clinical use.
In the past, the activation of prothrombin to thrombin was often brought about by the addition of calcium ions and thromboplastin (factor III). The thromboplastin would be derived from human or animal sources and was therefore a source of possible contamination of the final product.
U.S. Pat. No. 5,304,372 discloses a process for the preparation of a human thrombin concentrate from the PPSB fraction of plasma. The process comprises activation of the prothrombin in the PPSB fraction to produce thrombin by the addition of calcium chloride, followed by solvent-detergent treatment on the resulting product to inactivate viruses. According to U.S. Pat. No. 5,304,372 the solvent-detergent treatment cannot be carried out on the PPSB starting material because it would eliminate other factors such as phospholipids necessary for the activation reaction of prothrombin to thrombin.
EP-A-0,378,798 discloses a process for the preparation of thrombin which comprises absorbing factor II from plasma or plasma fractions onto a solid carrier,. activating the factor II on the solid carrier to thrombin and then eluting the thrombin.
U.S. Pat. No. 5,714,370 discloses the preparation of thrombin from virus-inactivated prothrombin using coagulatively active salts to effect the activation of the prothrombin to thrombin. The only method disclosed for the virus-inactivation of the prothrombin is heat treatment.
The 1993 Annual Report of the Dr. Karl Landsteiner Foundation on research at CLB (the Central Laboratory of the Netherlands Red Cross blood transfusion service) also reported on page 10 that activation of solvent-detergent treated prothrombin to form thrombin with calcium ions was only successful in the presence of added phospholipids.
It would be advantageous to provide a method for the preparation of thrombin from prothrombin which has undergone a solvent-detergent virus inactivation step to remove enveloped viruses. It would also be advantageous to avoid completely the use of other biological and particularly animal-derived products, for example thromboplastin, to activate the prothrombin, as such products may serve as a source of infectious viruses or other undesirable contaminants during the manufacturing process.